Extended Data Fig. 7: UVSSA and USP7 are required to prevent CSB degradation.
From: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4CSA-mediated degradation
a: CSB immunoblot of indicated HCT116 KO cells treated with 1 mM FA for 30 min and harvested at the indicated times. Tubulin was used as a loading control. This experiment was performed twice with similar results. b: Representative image of flow cytometry gating of CSB-mScarlet to determine CSB protein levels. Gating was used to exclude dead cells based on SCC-A/FSC-A plots and single cells were gated based on FSC-H/FSC-W and subsequently SSC-H/SSC-W. SSC- Side Scatter Channel, FSC- Forward Scatter Channel, A-Area, H-Height, W-Wight. c: Flow cytometry of CSB-mScarlet after a pulse of 1 mM FA for 30 min in the indicated cell lines. Cells were harvested at the indicated times and the fluorescent levels were determined by flow cytometry. Values were normalized to untreated levels and represent the average ±S.E.M. from 3 independent experiments. Unpaired two-tailed t-test. d: Flow cytometry of CSB-mScarlet after a pulse of 1 mM FA for 30 min in the presence of proteasome inhibitor MG132. Cells were harvested at the indicated times and the fluorescent levels were determined by flow cytometry. Values were normalized to untreated levels and represent the average ±S.E.M. from 3 independent experiments. Unpaired two-tailed t-test. e: Relative colony survival of U2OS KO cells treated with the indicated doses of FA for 1 hr and UV. Graph represents the mean with ±S.E.M. from 3 independent experiments. Unpaired two-tailed t-test at 6 J m-2 UV and 2 mM FA. f: Immunoblotting of cells after FA, stained for CSB and Tubulin. U2OS WT and USP7 KO cells were treated with 1 mM FA prior to harvesting at the indicated times. This experiment was performed twice with similar results. g: Immunoblot of siRNA transfected lysates of HeLa cells, stained with antibodies against CSB, USP7 and Tubulin. This experiment was performed twice with similar results. h: Relative colony survival of siRNA transfected HeLa cells treated with the indicated doses of FA for 1 hr. Graph represents the mean with ±S.E.M. from 3 independent experiments. Unpaired two-tailed t-test at 2.5 mM FA between UVSSA- and USP7-depleted cells. Source numerical data and unprocessed blots are available in source data.
